Surface micro-organism contamination assays

ABSTRACT

A receptacle and lid combination for molding solidified nutrient containing agar-agar with a flat face coplanar with the rim of the receptacle and keyed in the receptacle to resist withdrawal, for micro-organism contamination assays. The lid is formed with a flat base inside and the height of the circular wall on the receptacle is greater than the height of the circular wall on the lid to permit the receptacle to seat against the base of the lid and where the receptacle is formed with a vent and the lid is formed with projections from the wall near the base for a force fit with the receptacle.

United States Patent [72] Inventors David 1!. Land Horace Harding Expressway, Flushing, N.Y. l 1367; Stephen L. Bazil, 25 Catalpa Lane, Valley Stream, N.Y. 11581 [21] App]. No. 820,040 [22] Filed Apr. 24, 1969 [45] Patented Dec. 28, 1971 [54] SURFACE MICRO-ORGANISM CONTAMINATION ASSAYS 8 Claims, 7 Drawing Figs. [52] U.S. Cl. 195/139 [51] Int. Cl C12k 1/00 [50] Field of Search 195/103.5, 139 LE, 222 (189), 222 (540), 222 (460), 220 (44 [56] References Cited UNITED STATES PATENTS 2,361,992 11/1944 Cantor 195/139 LE Primary ExaminerA. Louis Monacell Assistant Examiner-Max D. Hensley AttorneysLouis A. Miller, Louis B. Applebaum and Arthur L. Bowers ABSTRACT: A receptacle and lid combination for molding solidified nutrient containing agar-agar with a flat face coplanar with the rim of the receptacle and keyed in the receptacle to resist withdrawal, for micro-organism contamination assays. The lid is formed with a flat base inside and the height of the circular wall on the receptacle is greater than the height of the circular wall on the lid to permit the receptacle to seat against the base of the lid and where the receptacle is formed with a vent and the lid is formed with projections from the wall near the base for a force fit with the receptacle.

SURFACE MICRO-ORGANISM CONTAMINATION ASSAYS The invention described herein may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment of any royalties thereon or therefor.

BACKGROUND OF THE INVENTION A technique for monitoring the quantity of micro-organisms that settle from the atmosphere in any particular outdoor or indoor location is to place at that location an upwardly directed open receptacle with shallow depth sterile solidified nutrient-containing clear agar-agar. The receptacle is transparent and is formed with a grid on the bottom delineating square centimeters. The depth of agar-agar is on the order of one-fourth inch. The period of exposure to the atmosphere is on the order of 12-24 hours. Micro-organisms that have deposited during that time cannot be seen even if the transparent receptacle and agar-agar is brightly illuminated. The receptacle is covered and is placed in an incubator set at temperature within the range 25 to 37 C. for about a day. The agar-agar remains solid during incubation; it liquifies at 60 C. and solidifies at 45 C. Individual micro-organisms captured on the surface of the agar-agar that thrive on the nutrient multiply into clusters or colonies during incubation. After the incubation period, the receptacle is illuminated from the rear. The colonies appear as dark spots to the naked eye. The assay is carried out by counting the number of dark spots in one or more of the squares.

A preferred modification of the above described technique that has been in common use eliminates the step of leaving an upwardly directed open receptacle in situ for hours and instead involves butting a surface of a thin agar-agar disk against a surface in a location to be monitored and withdrawing the agar-agar. Micro-organisms and minute particles on the monitored surface adhere to the agar-agar. The steps of culturing and assaying are otherwise the same as described above. The agar-agar disk for the modified technique is molded in a shallow transparent receptacle which is on the order of 2 inches in diameter and between one-eighth and one-fourth inch deep with centimeter square delineations in a grid pattern. A commercially marketed receptacle for the technique described is RODAC PLATE, a registered trademark of Falcon Plastics. Though termed a plate, this type product includes a shallow receptacle and a lid for that receptacle. This plate is designed to nest in another plate whereby several of them can be stacked in an incubator for space economy. In use, the shallow receptacle is filled with nutrient-containing agar-agar. The agar-agar is permitted to solidify with the lid in placeaThe surface of the solidified agar-agar is not coplanar with the rim; it has a concave meniscus. The Iidded receptacle containing the solidified agar-agar is brought to a flat surface to be monitored, the lid is removed, the receptacle is overturned and because of the meniscus needs to be pressed to flatten the concave surface of the agar-agar to force the face against the surface to be monitored. A percentage of the receptacles are ruined because the polystyrene or other resin of which the receptacle is formed cracks. In some other cases the agar-agar does not release from the monitor surface and instead is partly or completelypulled free of the receptacle when the receptacle is withdrawn. Because the pressure applied cannot be uniform and of the same magnitude for each disk, a variable is introduced limiting precision of the technique. Also this type of product can be used for growing aerobic micro-organisms only.

An object of this invention is to provide a receptacle and lid combination of the type described that overcomes the objections of the prior art, that is inexpensive, timesaving, takes up little room for growing aerobic and anaerobic bacteria and can be used for accurately determining surface contamination in hospitals, clinical laboratories, bacteriological research institutions, food-processing areas and food consumption areas, or any geographic area of BW defense program.

Other objects, advantages and novel features of the invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings wherein:

FIG. I is a top plan view ofa prior art receptacle,

FIG. 2 is a cross section of the receptacle of FIG. I with the lid in place.

FIG. 3 is a plan view of a receptacle in accordance with the principles of this invention,

FIG. 4 is sectional view of the receptacle shown in FIG. 3 with the lid in place,

FIG. 5 is a perspective view of another embodiment of receptacle,

FIG. 6 is a cross-sectional view of another embodiment of receptacle and lid combination according to the principles of this invention, and

FIG. 7 shows a structural arrangement of vent and handle for any of the receptacles.

SUMMARY OF THE INVENTION An agar-agar receptacle is formed to key the solidified agaragar against withdrawal when the receptacle is inverted. The agar-agar is solidified in the receptacle while inverted and seated on a flat surface or otherwise selectively shaped surface to form a flat or selected shape agar-agar face.

DESCRIPTION There is shown in FIGS. 1 and 2 a prior art type of receptacle l0 and lid 12 combination for monitoring surface areas for micro-organisms. The receptacle has a circular base with a short skirt l4 projecting from one face at the perimeter and a short circular ridge 16 of lesser diameter than the base projecting from the opposite face of the base. The lid 12 is circular, of larger diameter than the ridge 16 and deeper than the ridge to contain an air space and seats on the base around the ridge and of lesser diameter than the skirt for nesting into the skirt of another receptacle for stacking purposes. Several short projections are formed on the outer face of the lid whereby when the units are stacked in an incubator there is air circulation for uniform temperature.

In FIGS. 3 and 4 there is shown an embodiment of a receptacle 20 and lid 22 according to this invention. The receptacle includes a flat base 24 and a circular wall 26 projecting from one face of the base 24 and of smaller diameter than the base. Thin arcuate inwardly directed fins 28 project from the wall parallel to the base intermediate the base and the free end of the wall 26. The base 24 has a knockout 30. The lid 22 has a base 32 with an inner flat face and formed with a grid pattern of centimeter squares and a circular wall 34 slightly greater in diameter than the diameter of wall 26. The height of the wall 34 measured from the base 32 is slightly less than the height of wall 26 measured from the base 24. Three small integral nipplelike projections 36 are directed inwardly from the wall 34 near the base 32 and are of a size such that the lid and receptacle must be forced together for the receptacle to seat against the base 32 of the lid. If not forced together, the receptacle rests on the projections 36 when the lid 22 is underneath. The combination of receptacle and lid are packaged sterile.

In use, the combination shown in FIG. 4 is removed from the packaging and knockout 30 is displaced. The lid is substantially filled with nutrient-containing agar-agar and the receptacle is forced into the lid to seat against the bottom. An adhesive tape is used to cover the opening in the receptacle for shielding against contamination. The agar-agar is permitted to solidify. Because the free face of the agar-agar is solidified against a flat surface, the face is flat and coplanar with the rim of the receptacle. The receptacle and lid combination is taken to the surface to be monitored, separated, and the receptacle is butted against the surface. Since the face of the agar-agar is fiat, the entire face contacts the surface being monitored without pressure. If the surface to be monitored has a special shape, the base of the lid is made with a corresponding geometry. Then the receptacle is nested again in the lid and placed in the incubator. To culture anaerobic micro-organisms adhered to the agar-agar surface and not the aerobic micro-organisms, the receptacle and lid are pressed together, forcing out essentially all the air from between the face of the agar-agar and the inner face of the lid. Alternatively, the receptacle is nested in the lid on the nipplelike projections leaving an air space and permitting access of the atmosphere to the face of the agar-agar. After the incubation time, if the receptacle and lid were not previously pressed together, they are then pressed together. The combination is looked through toward a bright source. Spots per square in one or more of the squares are counted.

FIG. 5 shows another form of receptacle 38 which differs from the receptacle in FIG. 3 in that it includes a helical one-turn keying projection 40 instead of the keying projections 28 but is otherwise used in the same manner as is described. When molded, the receptacle 38 is unscrewed for removal; the mold for making the receptacle shown in FIG. 3 is more expensive.

The embodiment shown in FIG. 6 includes receptacle and lid 42 and 44 wherein the circular wall of each is frustoconical to serve the same purpose as the keying projections 28 in FIG. 3 and the keying projection 40 in FIG. 5, Instead of projection 36 in FIG. 4, projections 46 extend from the wall at the base of the lid. The receptacle 42 may be molded in one piece or may be made by bonding separate base and wall portions and then cementing or fusing them.

Each of the described receptacles may be formed with a knockout as described above, or with a hole that is sealed on the outer side with adhesive tape or with coaxial central cylindrical projection 50 for a fibrous plug 52 as shown in FIG. 7 for permitting passage of air and serving as a barrier against micro-organisms. Additionally, a handle 54 may be provided on the outer side of the receptacle.

Another feature included in this invention not shown on the drawings is to form the base of the receptacle so that the outer surface is flat and that the inner surface is slightly domed or coned. In other words the thickness of the base increases from its center outwardly to the circular wall of the receptacle. The purpose is to prevent the occlusion of bubbles of air around the inner surface of the base. By virtue of the domed or coned shape surface the air bubbles are driven to the center opening and do not occlude along the inner surface of the base.

We claim:

1. A receptacle and lid combination for nutrient-containing agar-agar for use in monitoring for micro-organisms comprismg a lid having a base with an essentially flat inside surface and a circular wall with at least three short equal height projections extending inwardly from the circular wall near said base, and

a receptacle having a base and a circular wall extending from the base of the receptacle and slightly longer than the circular wall of the lid and of outside diameter to fit into the circular wall of the lid to the projections and to force fit within the projections to seat against the base of the lid said base of said receptacle having a vent hole.

2. A receptacle and lid combination as defined in claim 1 wherein the circular wall of the receptacle is frustoconical and has its large diameter end at the base,

the circular wall of the lid is frustoconical and has its small diameter end at the base, and

the short equal height projections project inwardly from the wall at the base.

3. A receptacle and lid combination as defined in claim 1 wherein the circular wall of the receptacle is formed with at least one arcuate inwardly directed projection for keying agaragar that may be solidified in the receptacle.

4. A receptacle and lid combination as defined in claim 2 wherein the arcuately inwardly directed projection is a helical turn.

5. A receptacle and lid combination as defined in claim 2 wherein the circular wall of the receptacle is formed with four identical arcuate inwardly directed projections.

6. A receptacle and lid combination as defined in claim 1 wherein the center of the base of the receptacle is formed with a knockout.

7. A receptacle and lid combination as defined in claim 1 wherein the center of the base of the receptacle is formed with a coaxial cylinder for nesting a fibrous plug to permit passage of air but serving as a barrier to micro-organisms.

8. A receptacle and lid combination for nutrient containing agar-agar for use in monitoring for micro-organisms comprismg a lid having a base with an essentially flat inside surface and a frustoconical circular wall, and

a receptacle having a base and a frustoconical circular wall having its large diameter end at the base of the receptacle extending from the base of the receptacle and slightly longer than the circular wall of the lid and of outside diameter to fit into the circular wall of the lid to seat against the base of the lid, the base of the receptacle increasing in thickness from the center outwardly toward the circular wall said base of said receptacle having a vent hole. 

2. A receptacle and lid combination as defined in claim 1 wherein the circular wall of the receptacle is frustoconical and has its large diameter end at the base, the circular wall of the lid is frustoconical and has its small diameter end at the base, and the short equal height projections project inwardly from the wall at the base.
 3. A receptacle and lid combination as defined in claim 1 wherein the circular wall of the receptacle is formed with at least one arcuate inwardly directed projection for keying agar-agar that may be solidified in the receptacle.
 4. A receptacle and lid combination as defined in claim 2 wherein the arcuately inwardly directed projection is a helical turn.
 5. A receptacle and lid combination as defined in claim 2 wherein the circular wall of the receptacle is formed with four identical arcuate inwardly directed projections.
 6. A receptacle and lid combination as defined in claim 1 wherein the center of the base of the receptacle is formed with a knockout.
 7. A receptacle and lid combination as defined in claim 1 wherein the center of the base of the receptacle is formed with a coaxial cylinder for nesting a fibrous plug to permit passage of air but serving as a barrier to micro-organisms.
 8. A receptacle and lid combination for nutrient containing agar-agar for use in monitoring for micro-organisms comprising a lid having a base with an essentially flat inside surface and a frustoconical circular wall, and a receptacle having a base and a frustoconical circular wall having its large diameter end at the base of the receptacle extending from the base of the receptacle and slightly longer than the circular wall of the lid and of outside diameter to fit into the circular wall of the lid to seat against the base of the lid, the base of the receptacle increasing in thickness from the center outwardly toward the circular wall said base of said receptacle having a vent hole. 